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Results for “RNA Cap Analogs”

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PubMed2004

Phosphorylation states of translational initiation factors affect mRNA cap binding in wheat.

Phosphorylation of eukaryotic translational initiation factors (eIFs) has been shown to be an important means of regulating protein synthesis. Plant initiation factors undergo phosphorylation/dephosphorylation under a variety of stress and growth conditions. We have shown that recombinant wheat cap-binding protein, eIF(iso)4E, produced from E. coli can be phosphorylated in vitro. Phosphorylation of eIF(iso)4E has effects on m(7)G cap-binding affinity similar to those of phosphorylation of mammalian eIF4E even though eIF(iso)4E lacks an amino acid that can be phosphorylated at the residue corresponding to Ser-209, the phosphorylation site in mammalian eIF4E. The cap-binding affinity was reduced 1.2-2.6-fold when eIF(iso)4E was phosphorylated. The in vitro phosphorylation site for wheat eIF(iso)4E was identified as Ser-207. Addition of eIF(iso)4G and eIF4B that had also been phosphorylated in vitro further reduced cap-binding affinity. Temperature-dependent studies showed that DeltaH(degrees) was favorable for cap binding regardless of the phosphorylation state of the initiation factors. The entropy, however, was unfavorable (negative) except when eIF(iso)4E was phosphorylated and interacting with eIF(iso)4G. Phosphorylation may modulate not only cap-binding activity, but other functions of eukaryotic initiation factors as well.

Eukaryotic Initiation Factor-4E↗

PubMed1998

Internal and overall motions of the translation factor eIF4E: cap binding and insertion in a CHAPS detergent micelle.

The mRNA cap-binding protein eIF4E is the limiting factor in the eIF4F translation initiation complex, which mediates the binding of the 40S ribosome to the mRNA. 15N relaxation studies have been used to characterize the backbone dynamics of deuterated eIF4E in a CHAPS micelle for the apoprotein, the m7GDP-bound form, and the dinucleotide (m7GpppA)-bound form, as well as for CHAPS-free eIF4E. Large differences in overall correlation time between the CHAPS-free form (11.8 ns) and samples containing different concentrations of CHAPS (15.9-19.4 ns) indicate that eIF4E is embedded in a large micelle in the presence of CHAPS, with a total molecular weight in the range of 40-60 kDa. CHAPS seems to restrict the mobility of the a2-b3 and a4-b5 loops which are thought to be embedded in the micelle. No significant changes in overall mobility were seen between the m7 GDP-bound form, the m7GpppA-bound form, and the apoprotein. Amide hydrogen exchange data indicate the presence of slowly exchanging amides in two surface-exposed helices (a2 and a4), as well as the a4-b5 loop, indicating protection by the CHAPS micelle. The micelle covers the convex side of the protein away from the cap-binding site.

Cholic Acids↗

PubMed1979

Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.

Animals↗

PubMed1985

Isolation and characterization of factors from wheat germ that exhibit eukaryotic initiation factor 4B activity and overcome 7-methylguanosine 5'-triphosphate inhibition of polypeptide synthesis.

Three highly purified preparations (preparations I, II-1, and II-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and II-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations II-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.

Chromatography, Ion Exchange↗

PubMed1985

Cap-dependent RNA splicing in a HeLa nuclear extract.

We have studied the involvement of the 5' cap structure in the splicing of precursor mRNAs in a HeLa nuclear extract. We show that precursor mRNAs are spliced efficiently only when they possess a cap structure and that preincubation of a HeLa nuclear extract rendered the splicing reaction highly sensitive to inhibition by cap analogues. This sensitization was dependent on exogenous Mg2+ but not exogenous ATP or GTP. These results demonstrate that splicing in a nuclear extract is highly dependent on the cap structure, as was demonstrated for the splicing process in a HeLa whole-cell extract [Konarska, M. M., Padget, R. A. & Sharp, P. A. (1984) Cell 38, 731-736], and thus support the contention that cap recognition is an important feature of eukaryotic mRNA biogenesis.

Adenosine Triphosphate↗

PubMed1987

Preferential excision of the 5' proximal intron from mRNA precursors with two introns as mediated by the cap structure.

We have studied the effect of the 5' cap structure on the splicing of precursor mRNAs containing three exons and two introns within a single molecule in a HeLa nuclear extract. When a precursor mRNA was capped, the upstream intron was spliced out more efficiently than the downstream intron. The differential splicing reactions of the two introns are not due to differences in the intrinsic efficiency of splicing of each intron, since the preferential excision of the upstream intron was also observed when the positions of the two introns relative to the cap structure were reversed. When uncapped precursor mRNA was used as substrate, the downstream intron was spliced out appreciably, but splicing of the upstream intron was greatly reduced. Preincubation of the extract with cap analogues inhibited splicing of the upstream intron but not the downstream intron. Thus, the cap structure exerts its effect primarily on the 5' proximal intron.

HeLa Cells↗

PubMed1995

Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP.

After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule.

Alphavirus↗

PubMed1999

Identification of a nucleic acid binding domain in eukaryotic initiation factor eIFiso4G from wheat.

Higher plants have two complexes that bind the m7G-cap structure of mRNA and mediate interactions between mRNA and ribosomal subunits, designated eIF4F and eIFiso4F. Both complexes contain a small subunit that binds the 5'-cap structure of mRNA, and a large subunit, eIF4G or eIFiso4G, that binds other translation factors and RNA. Sequence-specific proteases were used to cleave native cap-binding complexes into structural domains, which were purified by affinity chromatography. We show here that eIFiso4G contains a central protease-resistant domain that binds specifically to nucleic acids. This domain spans Gln170 to Glu443 and includes four of the six homology blocks shared by eIFiso4G and eIF4G. A slightly shorter overlapping sequence, from Gly202 to Lys445, had no nucleic acid binding activity, indicating that the N-terminal end of the nucleic acid binding site lies within Gln170 to Arg201. The binding of the central domain and native eIFiso4F to RNA homopolymers and double- and single-stranded DNAs was studied. Both molecules had highest affinity for poly(G) and recognized single- and double-stranded sequences.

Amino Acid Sequence↗

PubMed2000

Stabilization of eukaryotic initiation factor 4E binding to the mRNA 5'-Cap by domains of eIF4G.

The eukaryotic cap-binding complex eIF4F is an essential component of the translational machinery. Recognition of the mRNA cap structure through its subunit eIF4E is a requirement for the recruitment of other translation initiation factors to the mRNA 5'-end and thereby for the attachment of the 40 S ribosomal subunit. In this study, we have investigated the mechanistic basis of the observation that eIF4E binding to the cap is enhanced in the presence of the large eIF4F subunit, eIF4G. We show that eIF4E requires access to both the mRNA 5'-cap and eIF4G to form stable complexes with short RNAs. This stabilization can be achieved using fragments of eIF4G that contain the eIF4E binding site but not the RNA recognition motifs. Full-length eIF4G is shown to induce increased eIF4E binding to cap analogues that do not contain an RNA body. Both results show that interaction of eIF4G with the mRNA is not necessary to enhance cap binding by eIF4E. Moreover, we show that the effect of binding of full-length eIF4G on the cap affinity of eIF4E can be further modulated through binding of Pab1 to eIF4G. These data are consistent with a model in which heterotropic cooperativity underlies eIF4F function.

Chromatography, Affinity↗

PubMedDate not supplied

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E.

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.

Animals↗

PubMed2005

Deaggregation of eIF4E induced by mRNA 5' cap binding.

All eukaryotic mRNAs contain a 5' terminal cap structure, which consists of 7-methylguanosine linked by a 5-5' triphosphate bridge to the first transcribed nucleoside (m7GpppN). Specific recognition of the cap by the eukaryotic initiation factor eIF4E plays a key role in regulation of translation initiation as a rate-limiting step. Using dynamic light scattering (DLS), the apo-form of murine eIF4E (33-217) was shown to aggregate. After addition of m7G7P, progressive deaggregation with the time of incubation in the presence of the cap analogue has been observed.

Animals↗

PubMed2001

Truncated initiation factor eIF4G lacking an eIF4E binding site can support capped mRNA translation.

Picornavirus proteases cleave translation initiation factor eIF4G into a C-terminal two-thirds fragment (hereafter named p100) and an N-terminal one-third fragment, which interacts with the cap-binding factor eIF4E. As the timing of this cleavage correlates broadly with the shut-off of host cell protein synthesis in infected cells, a very widespread presumption has been that p100 cannot support capped mRNA translation. Through the use of an eIF4G-depleted reticulocyte lysate system, we show that this presumption is incorrect. Moreover, recombinant p100 can also reverse the inhibition of capped mRNA translation caused either by m7GpppG cap analogue, by 4E-BP1, which sequesters eIF4E and thus blocks its association with eIF4G, or by cleavage of endogenous eIF4G by picornavirus proteases. The concentration of p100 required for maximum translation of capped mRNAs is approximately 4-fold higher than the endogenous eIF4G concentration in reticulocyte lysates. Our results imply that picornavirus-induced shut-off is not due to an intrinsic inability of p100 to support capped mRNA translation, but to the viral RNA outcompeting host cell mRNA for the limiting concentration of p100.

Animals↗

PubMed2002

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex.

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.

Amino Acid Sequence↗

PubMed1984

Synthesis of Gp4N and Gp3N compounds by guanylyltransferase purified from yeast.

Guanylyltransferase that catalyzes mRNA capping by the reaction, ppNpN + GTP----GpppNpN was purified from S. cerevisiae. The enzyme forms a nucleotidyl intermediate by phosphoamide linkage of GMP. Two guanylylated polypeptides of MR approximately 52,000 and 46,000 were obtained, the latter apparently by proteolysis of the larger component. Both forms transferred the covalently bound GMP to ppApG, yielding GpppApG. Dinucleoside tri- and tetraphosphates of the type Gp3N and Gp4N were also produced by using ribonucleoside 5'-di and triphosphates as acceptors. The purified yeast guanylyltransferase contained little or no RNA 5'-triphosphatase or methyltransferase.

Molecular Weight↗

PubMed1984

Chemical synthesis of the 5'-terminal part bearing cap structure of messenger RNA of cytoplasmic polyhedrosis virus (CPV): m7G5'pppAmpG and m7G5'pppAmpGpU.

The 5'-terminal structures of mRNA bearing the so-called 'cap' from cytoplasmic polyhedrosis virus (CPV), m7G5' pppAmpG and m7G5' pppAmpGpU, were first chemically synthesized. S,S-Di(4-methoxyphenyl) N6-benzoyl-2'-O-methyladenosine 5'-phosphorodithioate ((ArS) 2pAbmz) was prepared by phosphorylation of the 5'-hydroxyl group of N6-benzoyl-2'-O-methyladenosine with S,S-di(4-methoxyphenyl) phosphorodithioate by TPS. By the triester approach using (ArS) 2pAbmz as starting material, the protected dinucleotide and trinucleotide bearing 5'-phosphate group were synthesized. The protective groups of the dinucleotide and trinucleotide were removed to obtain pAmpG and pAmpGpU, respectively. By the reaction of a capping agent ((PhS) ppm7G) with pAmpG and pAmpGpU in the presence of silver nitrate or iodine. The 5'-terminal structure of the messenger RNA strand of CPV which was labelled isotopically, was confirmed completely as m7G5' pppAmGpU by cochromatography with the materials chemically synthesized here.

Chemical Phenomena↗

PubMed1988

A selective recognition mode of a nucleic acid base by an aromatic amino acid: L-phenylalanine-7-methylguanosine 5'-monophosphate stacking interaction.

The conformation of 7-methylguanosine 5'-monophosphate (m7GMP) and its interaction with L-phenylalanine (Phe) have been investigated by X-ray crystallographic, 1H-nuclear magnetic resonance, and energy calculation methods. The N(7) methylation of the guanine base shifts m7GMP toward an anti--gauche, gauche conformation about the glycosyl and exocyclic C(4')-C(5') bonds, respectively. The prominent stacking observed between the benzene ring of Phe and guanine base of m7GMP is primarily due to the N(7) guarternization of the guanine base. The formation of a hydrogen bonding pair between the anionic carboxyl group and the guanine base further stabilizes this stacking interaction. The present results imply the importance of aromatic amino acids as a hallmark for the selective recognition of a nucleic acid base.

Crystallography↗

PubMed1979

Specific inhibition of capped mRNA translation in vitro by m7G5'pppp5'G and m7G5'pppp5'm7G.

A unique set of diguanosine cap analogues containing a 5'-5' tetraphosphate linkage instead of the normal triphosphate was synthesized by chemical methylation of G5'pppp5'G. Both 7-methylguanosine products, m7G5'pppp5'G and m7G5'pppp5'm7G, acted as potent inhibitors of capped brome mosaic virus (BMV) RNA translation in the homologous wheat germ protein synthesis system. Inhibition of in vitro protein synthesis required the presence of the 7-methyl group on guanosine and was specific for capped mRNA. In comparison with the partial cap analogue, m7GTP, the methylated diguanosine tetraphosphate structures were 25-50 fold more potent inhibitors of in vitro protein synthesis. Analysis of the in vitro translation products of the four species of BMV RNA showed a differential sensitivity to inhibition by m7G5'pppp5'm7G.

Methylation↗

PubMed1987

In vitro splicing of a chicken delta-crystallin pre-mRNA in a mammalian nuclear extract.

An in vitro splicing system was constructed using portions of chicken delta-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25% of the pre-mRNA was precisely spliced at 30 degrees C in 2 h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human beta-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.

Animals↗